Treatment of diseases related to hyperactivity of the complement system

ABSTRACT

Raising the level of Factor I above physiological levels can be used to treat diseases in which the underlying pathology is linked to overactivity of the C3b-feedback cycle and the generation and pro-inflammatory effects of iC3b. Methods, agents, and compositions for treatment of such diseases are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/753,193, filed Jun. 29, 2015, which will issue as U.S. Pat. No.9,782,460 on Oct. 10, 2017, which is a continuation of U.S. patentapplication Ser. No. 13/256,311, filed Dec. 5, 2011, now U.S. Pat. No.9,066,941, issued Jun. 30, 2015, which was a national phase entry under35 U.S.C. § 371 of International Patent Application PCT/GB2010/000465,filed Mar. 12, 2010, designating the United States of America andpublished in English as International Patent Publication WO 2010/103291A2 on Sep. 16, 2010, which claims the benefit under Article 8 of thePatent Cooperation Treaty to Great Britain Patent Application Serial No.0904427.2, filed Mar. 13, 2009, the disclosure of each of which ishereby incorporated herein in its entirety by this reference.

TECHNICAL FIELD

This application relates to agents, compositions and methods for theprevention, treatment, or amelioration of diseases in which theunderlying pathology is linked to overactivity of the C3b-feedback cycleand the generation and pro-inflammatory effects of iC3b, a product ofthe activation of the complement system.

BACKGROUND

The complement system comprises a set of about 30 proteins that may belocated in the fluid phase (generally, in plasma) or on the surfaces ofcells in which the proteins are expressed. The system serves severalimportant biological functions related to both innate and adaptiveimmunity and is phylogenetically ancient. It must recognize foreign(non-self) entities, react to them in a highly amplifiable way in orderto trigger an effective defensive biological response and yet keep thesystem under tight control to avoid collateral (“self”) damage. Thecomponents may be grouped into eight functional classes:

-   -   1. Recognition molecules that bind to pathogen-associated        molecular patterns not found in the host organism (e.g.,        bacterial carbohydrate) or to antigens defined through previous        encounter or as part of the pre-existing immunoglobulin        repertoire. An example is Mannan-binding lectin (MBL).    -   2. Central non-enzymatic effector proteins that undergo        combinations and transformations critical to the system (e.g.,        C3, C4).    -   3. Central enzymatic effectors that also participate in these        processes (e.g., C1 esterase, C2, Factor B).    -   4. Amplifiers of the activation processes either enzymatic        (Factor D) or non-enzymatic (Properdin).    -   5. Terminal components that do not undergo feedback processes        but give rise to the final cytolytic membrane attack complex        (components C5 to C9).    -   6. Soluble negative regulators either enzymatic (Factor I) or        non-enzymatic (e.g., Factor H, C4bp).    -   7. Negative regulators located on the surfaces of cells that        they protect against attack by the endogenous complement system        (e.g., CR1, DAF, MCP, CD59).    -   8. Receptors that permit cellular signaling by products of        complement activation, thus linking the process to other immune        and cell regulatory functions (e.g., CR2, CR3, C3a, C5a        receptor).

Although the general functions of the complement system have been knownfor several decades, the details of the processes, their evolution andregulation and the relationship of the system to cellular immunity aremore recent scientific achievements. These developments can besummarized as follows (from P. J. Lachmann (1979), An evolutionary viewof the complement system, Behring Inst. Mitt. no. 63 25-37):

-   -   1. The archaeo-complement system probably consisted of a form of        the C3 protein alone—this was cleaved by microbial proteases        into C3a and C3b. The small C3a fragment is a chemotactic        factor. The large fragment, C3b, acquires briefly the capacity        to attach covalently to the microbe following cleavage of its        thiol ester group. Bound C3b acts as an “opsonization marker”        marking the microbe for destruction by phagocytic cells. The        recent determination of the crystal structures of C3 and C3b        (B. J. C. Janssen et al., Nature 444:213-6, 2006) has shown how        the thiol ester is buried within the multi-domain C3 molecule        and is exposed in C3b following a major rearrangement of the        domains.    -   2. The complement system achieved a primary level of        amplification through addition of the protease Factor B, which        mediates positive feedback by combining with the C3b product to        form a complex, C3bB that, after cleavage by microbial or other        proteases, forms a convertase, C3bBb, capable of activating more        C3.    -   3. This system was further amplified to create the current        “alternative pathway” or “C3b feedback” loop by recruiting        Factor D, a protease probably used for other purposes and that        is only active when its substrate, Factor B, is complexed to        C3b. FD resists all plasma protease inhibitors and allows the        C3b feedback cycle to function in plasma.    -   4. Together with Properdin, these components created the basis        of the rapid and highly amplified response to external        pathogens. Addition of the lectin and “classical”        (antibody-triggered) systems followed.    -   5. The corresponding control system to prevent excessive C3b        generation in higher vertebrates with pumped blood circulation        was provided by a soluble plasma protease (FI), which catalyzes        the cleavage of C3b to iC3b (the “First clip”). Factor I will        cleave C3b only when this is complexed to a molecule with        “Factor I cofactor activity.” In plasma the principal molecule        with this property is Factor H.    -   6. iC3b cannot participate in convertase formation and, hence,        amplification, but it can function as a powerful        pro-inflammatory agent through interaction with complement        receptor type 3 (CR3, CD11b/CD18), an integral membrane protein        found on neutrophils and monocytes that engages iC3b, a reaction        enhanced by also binding microbial carbohydrate (e.g., beta        glucan, (Y. Xia, V. Vetvicka, J. Yan, M. Hanikýrová, T. Mayadas,        and G. D. Ross (1999), the beta-glucan-binding lectin site of        mouse CR3 (CD11b/CD18) and its function in generating a primed        state of the receptor that mediates cytotoxic activation in        response to iC3b-opsonized target cells, J. Immunol. 162        (4):2281-90).    -   7. iC3b can be further broken down by Factor I (FI) and the        membrane-bound cofactor CR1 (CD35) in a so-called “Second clip,”        which yields C 3 d,g-a C3 fragment, which is not        pro-inflammatory (it does, however, have an effect on the        adaptive immune system through stimulation of specific antibody        production through interaction with CR2) and C3c.

These processes are illustrated in FIG. 1. This shows an outline of theactivation of the complement system initiated by recognition events atthe beginning of the pathways and amplified by the C3b amplificationloop—the generation and deactivation of iC3b. The C3b amplification loopis also shown in FIG. 2. This amplification loop is a balance betweentwo competing cycles both acting on C3b: the C3 feedback cycle thatenhances amplification, and the C3 breakdown cycle that down-regulatesit. It is solely the balance between their rates of reaction on whichamplification depends. The C3 breakdown cycle generates iC3b as itsprimary reaction product. iC3b, through its reaction with the leukocyteintegrins (and complement receptors) CR3 (CD11b/CD18) and CR4(CD11c/CD18), is the most important mechanism by which complementmediates inflammation.

BRIEF SUMMARY

The disclosure is derived from the understanding that the geneticpredisposing factors for several inflammatory diseases all serve toenhance the activity of the C3b feedback cycle, thereby creating apro-inflammatory phenotype of which the formation of iC3b and itsreaction with CR3 is a critical component.

According to the disclosure, there is provided a method of preventing,treating, or ameliorating a disease associated with overactivity of thecomplement C3b feedback cycle, which comprises increasing the level ofC3b-inactivating and iC3b-degradation activity in a subject to a levelthat exceeds a normal level of C3b-inactivating and iC3b-degradationactivity.

According to the disclosure, there is also provided an agent (or agents)with C3b-inactivating and iC3b-degradation activity for use as amedicament.

According to the disclosure, there is further provided an agent (oragents) with C3b-inactivating and iC3b-degradation activity for use inthe prevention, treatment, or amelioration of a disease associated withoveractivity of the complement C3b feedback cycle, at a dosage thatincreases the level of C3b-inactivating and iC3b-degradation activity ina subject to a level that exceeds a normal level of C3b-inactivating andiC3b-degradation activity.

According to the disclosure, there is further provided use of an agent(or agents) with C3b-inactivating and iC3b-degradation activity in themanufacture of a medicament for the prevention, treatment, oramelioration of a disease associated with overactivity of the complementC3b feedback cycle, at a dosage that increases the level ofC3b-inactivating and iC3b-degradation activity in a subject to a levelthat exceeds a normal level of C3b-inactivating and iC3b-degradationactivity.

The term “complement C3b feedback cycle” is used herein to refer to apositive feedback cycle acting through C3b (the major product of C3cleavage), which interacts with factors B and D of the alternativepathway to form a C3-cleaving enzyme.

“Overactivity of the complement C3b feedback cycle,” means that there isincreased formation of the C3-cleaving enzyme compared with a normalsubject, with consequent increased turnover of C3 and components of thealternative pathway. The turn-over of C3 can be measured in-vivo using¹²⁵I-labelled C3. Alternatively turn-over of C3 can be measuredindirectly in-vitro by determining the rate of C3 conversion to iC3bwhen a small complement activating stimulus is given (as described in P.J. Lachmann and L. Halbwachs (1975), the influence of C3b inactivator(KAF) concentration on the ability of serum to support complementactivation, Clin. Exp. Immunol. 21:109).

Preferably, the disease is associated with an ongoing predisposition tooveractivity of the C3b feedback cycle. This means that whenever thecomplement system is activated during the course of the disease, thereis an overactivity of the C3b feedback cycle. This is distinguished overdiseases in which there may be a temporary susceptibility tooveractivity of the C3b feedback cycle, for example, during a particularphase of the disease. Factor I and Factor H have been reported to bedecreased in the pre or early phases of the exacerbation stage, but notduring most of the regression stage of systemic lupus erythematosus(SLE).

Preferably, the disease is an inflammatory disease.

Preferably, the disease is not an autoimmune disease, especially SLE,rheumatoid arthritis or glomerulonephritis.

Examples of diseases that may be prevented, treated, or ameliorated bymethods of the disclosure are Age-related Macular Degeneration (AMD),atypical hemolytic uremic syndrome (aHUS), membranoproliferativeglomerulonephritis Type 2 (MPGN2), atherosclerosis (in particular,accelerated atherosclerosis), chronic cardiovascular disease, andAlzheimer's disease (particularly Alzheimer's disease in a subjectcarrying an ApoE4 allele, i.e., a subject who is heterozygous orhomozygous for the ApoE4 allele).

The subject may have a genetic predisposition to the disease with orwithout a family history of the disease. Accordingly, methods of thedisclosure may further comprise determining whether the subject has agenetic predisposition to the disease or a family history of thedisease, and administering appropriate prophylaxis or therapy dependingon the result of the determination.

Examples of genetic predispositions to diseases associated withoveractivity of the complement C3b feedback cycle (preferably, anongoing predisposition to overactivity of the C3b feedback cycle) are: amutation in Factor H that reduces its ability to function as a Factor Icofactor compared with wild-type Factor H; a mutation in Factor H thatreduces its binding to C3b compared with wild-type Factor H; homozygousFactor H deficiency; a mutation in membrane cofactor protein (MCP) thatreduces its function compared with wild-type MCP; heterozygous Factor Ideficiency; a gain-of-function mutation in Factor B; or a C3F allotype.

All the predisposing alleles share the property of enhancing theactivity of the C3b amplification loop by either upregulating the C3bfeedback cycle or downregulating the C3b breakdown cycle. They all,therefore, promote a hyperinflammatory complement phenotype. This willproduce its effects by increasing the production of C5a and of themembrane attack complex and, most importantly, by the increasedproduction of iC3b, which, through its reaction with CR3 (CD11b/CD18)and CR4 (CD11c/CD18) on neutrophils, monocytes and NK cells, providescomplement's most powerful pro-inflammatory mechanism.

The term “agent with C3b-inactivating and iC3b-degradation activity” isused herein to mean an agent with serine protease activity that is ableto catalyze the cleavage of C3b to iC3b (the “First Clip”) anddegradation of iC3b (the “Second Clip”). The agent may require one ormore cofactors in order to catalyze these reactions. For example, FactorH may be required for the First Clip, and CR1 may be required for theSecond Clip.

A preferred example of the agent is Factor I. However, other agents withfunctional equivalence to Factor I may alternatively be used, such asfragments or derivatives of Factor I that retain C3b-inactivating andiC3b-degradation activity. The Factor I, or fragment or derivative, maybe plasma-derived Factor I, or recombinant Factor I, or fragment orderivative. Preferably, the Factor I is of the same species as thesubject.

The skilled person will appreciate that derivatives of Factor I thatretain C3b-inactivating and iC3b-degradation activity may be prepared byproviding a protein comprising a sequence that differs from nativeFactor I sequence by one or more conservative amino acid substitutions,and/or by deletion of one or more amino acid residues. Preferably, suchFactor I derivatives retain at least 60% amino acid identity across theentire length of the sequence with native Factor I. More preferably, aFactor I derivative retains at least 70%, 80%, 90%, or 95% amino acididentity across the entire length of the sequence with native Factor I.

It is also envisaged that the agent could be provided in a form thatrequires modification (for example, prior to administration, or in vivo)to provide the C3b-inactivating and iC3b-degradation activity. It isalso envisaged that the C3b-inactivating and iC3b-degradation activitycould each be provided by separate agents, which may be administeredtogether or separately (for example, sequentially).

One or more agents with C3b-inactivating and iC3b-degradation activitymay be administered. Preferably, the agent(s) is(are) not administeredwith Factor H.

Preferably, the subject has a normal level of C3b-inactivating andiC3b-degradation activity provided by the subject's Factor I. A normallevel is regarded to be in the range 30-40 μg/ml Factor I in thesubject's plasma.

Measurement of plasma Factor I can be determined using conventionalmethods, for example, antigenically, using radial immunodiffusion,“rocket” electrophoresis, or nephelometry or functionally, usingconglutinin (Lachmann and Muller-Eberhard, 1968, J. Immunol. 100:691),or a hemolytic inhibition assay (Lachmann, Nicol and Aston, 1973,Immunochem. 10:695).

Preferably, the level of C3b-inactivating and iC3b-degradation activityin the subject's plasma is increased by at least 10% above the normallevel, preferably for a period of at least one to two weeks. However,the level of activity in the subject's plasma is preferably increased byno more than 50%, preferably no more than 25% above the normal level.

Preferably, the level of activity is increased by administering anagent(s) with C3b-inactivating and iC3b-degradation activity to thesubject. The agent is preferably used at a dosage for increasing thelevel of activity in the subject's plasma by at least 10% above thenormal level, preferably for a period of at least one to two weeks.However, the dosage preferably increases the level of activity in thesubject's plasma by no more than 50%, preferably no more than 25% abovethe normal level.

The agent (preferably Factor I) is preferably used at a dosage up to 20mg/kg, for example, 0.001 or 0.05 mg/kg to 20 mg/kg, more preferablyless than 6 mg/kg, for example, 0.001 or 0.05 mg/kg to less than 6mg/kg, more preferably up to 1.5 mg/kg, for example, 0.001, 0.05, or 0.2mg/kg to 1.5 mg/kg or 0.001, 0.05, or 0.2 mg/kg to 1 mg/kg, or less than1 mg/kg, for example, 0.001, 0.05, or 0.2 mg/kg to less than 1 mg/kg.Preferred doses for human subjects are up to 250 mg, for example, 6.5 or10 mg to 250 mg, preferably less than 50 mg, for example, from 6.5 or 10mg to less than 50 mg, or 10-20 mg. For systemic administration, theagent may preferably be administered at a dosage up to 250 mg, forexample, from 6.5 or 10 mg to 250 mg, preferably from 6.5 or 10 mg toless than 50 mg, for example, 10-20 mg. For intravenous or intramuscularadministration, the agent may preferably be administered at a dosage of0.05 to 20 mg/kg, for example, 0.05 to less than 6 mg/kg, or 0.05 toless than 1 mg/kg. For intraocular administration the agent may beadministered a dosage of 0.001 to 1 mg/eye Factor I.

The level of activity may be increased by administering C3b-inactivatingand iC3b-degradation activity at least once per month, or at least onceper day.

It will be appreciated that the appropriate frequency of administrationof C3b-inactivating and iC3b-degradation activity will depend on manyfactors, including the route of administration, the type of disease andthe severity, stage of the disease, the plasma half life of theC3b-inactivating and iC3b-degradation activity in the preparation (theplasma half life of Factor I is believed to be approximately one week),the background factor I levels in the patient, the desired steady-stateprotein concentration level, the influence of any therapeutic agentsused in combination with the treatment method of the disclosure, theage, health, and sex of the subject. In many cases, it will beappropriate to monitor progress of the disease to determine the effectof the treatment, and based on the results determine whether or nottreatment should be continued, and the appropriate frequency of thetreatment.

If the disease is an acute disease, such as atypical hemolytic uremicsyndrome (aHUS), C3b-inactivating and iC3b-degradation activity ispreferably administered if the subject is determined to be sufferingfrom an infection or a fever. Progress of the aHUS may be monitored bythe blood platelet count, the presence of red cell fragments in theblood, the presence of raised levels of lactate dehydrogenase (LDH) inthe blood, or the concentration of urea or creatinine in the blood,and/or by the enumeration in urine of red blood cells, white bloodcells, and casts and measurement of urinary protein.

If the disease is a chronic disease, such as Age-related MacularDegeneration (AMD), C3b-inactivating and iC3b-degradation activity ispreferably administered every 2 to 4 weeks. Progress of AMD may bemonitored by determining the extent of drusen formation (deposits thataccumulate beneath the retinal pigmented epithelium). If the extent ofdrusen formation is reduced, or if the rate of drusen formation isreduced by the treatment, then the frequency of administration of theC3b-inactivating and iC3b-degradation activity may also be reduced.

Alternatively, or additionally the progress of AMD may be monitored bymeasuring in vivo the amount of C3 fragments bound in the retina. Thismay be achieved, for example, by administering a binding agent thatbinds to iC3b but not to C3 or C3b. Preferably, the binding agent is amonoclonal antibody or a fragment or derivative thereof that retainsbinding specificity for iC3b. It will be appreciated that the monoclonalantibody or fragment or derivative should not induce an adverse immunereaction in the subject to whom it is administered. Thus, it may bedesirable to use a humanized monoclonal antibody, or a derivative suchas a single chain Fv, where the subject is a human subject.

Preparation of a monoclonal antibody that binds iC3b (but not native C3or C3b) is described in Lachmann et al. (Immunology, 1980, 41(3):503-515—Clone 9). This antibody is available commercially fromHycult Biotechnology b.v. (Catalog no. HM2199, Monoclonal antibody tohuman C3g, clone 9, also known as YB2/90-5-20; the antibody recognizesiC3b, C3dg and C3g in plasma, but does not recognize C3 or C3b). Asingle chain Fv derived from YB2/90-5-20 may be used as a suitablebinding agent for administration to a human subject.

The binding agent should be labeled so that it can be detected. In apreferred embodiment, the binding agent is labeled with fluorescein.After intravenous injection, this will stain deposits in the eye, whichcan then be quantitated by fluorography.

It will be appreciated that binding agents that bind to iC3b, but not toC3 or C3b, may be used for the diagnosis of an inflammatory lesion inthe eye, for example, as a result of Age-related Macular Degeneration(AMD).

According to the disclosure, there is provided a method for diagnosingwhether a subject has an inflammatory lesion in an eye, which comprisesadministering a binding agent that binds to iC3b, but not to C3 or C3b,to the subject, and determining whether the binding agent binds to theretina in the eye of the subject.

Presence of binding agent bound at the retina in the eye of the subjectindicates that the subject has an inflammatory lesion in that eye.

According to the disclosure, there is further provided a method formonitoring progression of an inflammatory lesion in an eye of a subject,which comprises administering to the subject a binding agent that bindsto iC3b, but not to C3 or C3b, at a first point in time and at asubsequent second point in time, and determining the amount of bindingagent that binds to the retina in an eye of the subject at the secondpoint in time relative to the first point in time.

An increase in the amount of binding agent that binds to the retina atthe second point in time relative to the first point in time indicatesthat the inflammatory lesion has progressed in the subject. No change inthe amount of binding agent that binds to the retina at the second pointin time relative to the first point in time indicates that theinflammatory lesion has not progressed in the subject. A decrease in theamount of binding agent that binds to the retina at the second point intime relative to the first point in time indicates that the inflammatorylesion has regressed in the subject.

It will be appreciated that methods of the disclosure for monitoringprogression of an inflammatory lesion may be used to determine theeffectiveness of treatment of the inflammatory lesion in the subject.

There is also provided, according to the disclosure, use of a bindingagent that binds to iC3b, but not to C3 or C3b, to diagnose aninflammatory lesion in an eye of a subject, or to monitor theprogression of an inflammatory lesion in an eye of a subject.

The disclosure further provides a kit for diagnosing whether a subjecthas an inflammatory lesion, or for monitoring the progression of aninflammatory lesion, in an eye of the subject, which comprises a bindingagent that binds to iC3b, but not to C3 or C3b, and instructions fordiagnosing whether the subject has an inflammatory lesion, or formonitoring the progression of an inflammatory lesion, in an eye of thesubject using the binding agent. The instructions may describeadministration of the binding agent to the subject and/or how todetermine whether the binding agent binds to the retina in an eye of thesubject.

The binding agent may be labeled with a label that allows detection ofthe binding agent at the retina. A suitable label is a fluorescentlabel, for example, a fluorophore such as fluorescein.

The binding agent may be administered systemically, preferablyintravenously, to the subject.

There is also provided, according to the disclosure, a compositioncomprising a binding agent that binds to iC3b, but not to C3 or C3b,together with a pharmaceutically acceptable carrier, excipient, ordiluents. The composition is preferably suitable for systemic,preferably intravenous administration.

The binding agent is preferably a monoclonal antibody or a fragment orderivative of a monoclonal antibody.

According to the disclosure, there is also provided a fragment orderivative of a monoclonal antibody that binds to iC3b, but not to C3 orC3b. Preferably, the derivative is a single chain Fv (scFv), forexample, an scFv of YB2/90-5-20. Preferably, the fragment or derivativeis labeled, suitably with a fluorescent label, for example, afluorophore such as fluorescein.

Such fragments or derivatives may be used as binding agents in methodsor kits of the disclosure for the diagnosis of an inflammatory lesion,or for monitoring the progression of an inflammatory lesion, in an eyeof a subject.

Methods and kits of the disclosure for the diagnosis of an inflammatorylesion, or for monitoring the progression of an inflammatory lesion, inan eye of a subject may be used for the diagnosis of AMD, or formonitoring the progression of AMD, in the subject.

In preferred aspects of the disclosure, the subject is a human subject.However, it may alternatively be desired to ameliorate, treat, orprevent (or diagnose or monitor progression of) the disease in non-humananimals, such as domestic pets.

There is further provided, according to the disclosure, a unit dosecomprising an agent or agents with C3b-inactivating and iC3b-degradationactivity for administration to a subject.

There is also provided, according to the disclosure, a pharmaceuticalcomposition in unit dose form, which comprises an agent or agents withC3b-inactivating and iC3b-degradation activity, and a pharmaceuticallyacceptable carrier, excipient, or diluent for administration to asubject.

There is also provided, according to the disclosure, a pharmaceuticalcomposition, which comprises an agent or agents with C3b-inactivatingand iC3b-degradation activity, and a pharmaceutically acceptablecarrier, excipient, or diluent for administration to a subject.

There is further provided, according to the disclosure, a unit dose for,or adapted for intraocular administration, which comprises an agent oragents with C3b-inactivating and iC3b-degradation activity.

There is further provided, according to the disclosure, a pharmaceuticalcomposition, preferably in unit dose form, for or adapted forintraocular administration, which comprises an agent or agents withC3b-inactivating and iC3b-degradation activity, and a pharmaceuticallyacceptable carrier, excipient, or diluent.

There is further provided, according to the disclosure, a unit dose for,or adapted for systemic administration, which comprises an agent oragents with C3b-inactivating and iC3b-degradation activity. The unitdose for, or adapted for, systemic administration should be sterile, andfree of pyrogens and viruses.

There is further provided, according to the disclosure, a pharmaceuticalcomposition, preferably in unit dose form, for or adapted for systemicadministration, which comprises an agent or agents with C3b-inactivatingand iC3b-degradation activity, and a pharmaceutically acceptablecarrier, excipient, or diluent.

Systemic administration is preferably intravenous or intramuscularadministration.

A unit dose or composition of the disclosure may comprise up to 250 mg,for example, from 1 or 10 mg to 250 mg, preferably less than 50 mg, forexample, from 1 or 10 mg to less than 50 mg, preferably 10-20 mg, of anagent or agents with C3b-inactivating and iC3b-degradation activity.

There is further provided, according to the disclosure, a unit dose orcomposition of the disclosure for, or adapted for, administration to ahuman subject.

A unit dose or composition of the disclosure may be in solid form,preferably lyophilized form.

Preferably, the agent of the unit dose or composition of the disclosureis Factor I, or a fragment or derivative of Factor I that retainsC3b-inactivating and iC3b-degradation activity. The Factor I, orfragment or derivative, may be plasma-derived Factor I, or recombinantFactor I, or fragment or derivative.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the disclosure are described below withreference to the accompanying drawings in which:

FIG. 1 shows an outline of the activation of the complement systeminitiated by recognition events at the beginning of the pathways andamplified by the C3b amplification loop—the generation and deactivationof iC3b and the dependence of those events on Factor I, Factor Icofactors and Factor H is noted;

FIG. 2 shows the C3b amplification loop, which is a balance between twoseparate and competing pathways—the C3b feedback cycle and the C3bbreakdown cycle; and

FIG. 3 shows the effect of increased factor I (FI) concentration on C3conversion by inulin and by aggregated IgG.

DETAILED DESCRIPTION

In a preferred embodiment, the disclosure provides for a therapy that byraising Factor I concentration reduces the activity of the C3b-feedbackcycle.

1. Disease-Associations of Aberrant Complement Activation

Recent gene-association studies have shown a strong influence ofcomplement system components in several diseases and conditions, withmost attention having focused on Age-related Macular Degeneration (AMD)and atypical hemolytic uremic syndrome (aHUS), the latter having, sofar, been more extensively studied.

Thus:

Mutations in Factor H that give reduced function as a Factor I co-factoror reduce its binding to C3b predispose to AMD and aHUS. HomozygousFactor H deficiency also has been associated with aHUS as haveautoantibodies to Factor H.

Mutations in the cell-bound complement control protein, membranecofactor protein (MCP or CD46) that give reduced function predispose toaHUS.

(The extensive literature on these associations is reviewed in A.Richards, D. Kavanagh, and J. P. Atkinson (2007), Inherited ComplementRegulatory Protein Deficiency Predisposes to Human Disease in AcuteInjury and Chronic Inflammatory States, The Examples of Vascular Damagein Atypical Hemolytic Uremic Syndrome and Debris Accumulation inAge-Related Macular Degeneration, Adv. Immunol. 96:141-77.)

Heterozygous deficiency of Factor I (but not homozygous (total) Factor Ideficiency) is associated with aHUS (D. Kavanagh, A. Richards, M. Noris,R. Hauhart, M. K. Liszewski, D. Karpman, J. A. Goodship, V.Fremeaux-Bacchi, G. Remuzzi, T. H. Goodship, and J. P. Atkinson (2008),characterization of mutations in complement factor I (CFI) associatedwith hemolytic uremic syndrome, Mol. Immunol. 95-105).

Gain-of-function mutations in complement factor B are associated withatypical haemolytic uraemic syndrome (E. Goicoechea de Jorge, C. L.Harris, J. Esparza-Gordillo, L. Carreras, E. A. Arranz, C. A. Garrido,M. Lopez-Trascasa, P. Sanchez-Corral, B. P. Morgan, and S. Rodriguez deCordoba (2007), mutations in Factor B that increase its function as aC3-convertase predispose to aHUS, Proc. Natl. Acad. Sci. U.S.A. 104(1):240-5).

The C3F allotype is associated with an increased risk of AMD (J. R.Yates, T. Sepp, B. K. Matharu, J. C. Khan, D. A. Thurlby, H. Shahid, D.G. Clayton, C. Hayward, J. Morgan, A. F. Wright, A. M. Armbrecht, B.Dhillon, I. J. Deary, E. Redmond, A. C. Bird, and A. T. Moore (2007),complement C3 variant and the risk of age-related macular degeneration,N. Engl. J. Med. 357:553-61). There is also a long-standing finding thatC3F carries an increased risk of atherosclerotic vascular disease (H.Sorensen and J. Dissing (1975), association between the C3F gene andatherosclerotic vascular diseases, Hum. Hered. 25 (4):279-83).

C3F augments the C3b-feedback cycle by forming a more activeC3-convertase (C. Harris and B. P. Morgan personal communication).

In mice, membranoproliferative glomerulonephritis Type 2 (MPGN2, a renalinflammatory condition), occurs spontaneously in Factor H knockout (k/o)mice and results in consumption of C3, and iC3b deposition in glomeruli.If such k/o mice are then made to express a mutant form of FHfunctionally equivalent to the FH mutant associated with aHUS in man,they develop aHUS but not MPGN2 (M. C. Pickering, E. G. de Jorge, R.Martinez-Barricarte, S. Recalde, A. Garcia-Layana, K. L. Rose, J. Moss,M. J. Walport, H. T. Cook, S. R. de Cordoba, and M. Botto (2007),spontaneous hemolytic uremic syndrome triggered by complement factor Hlacking surface recognition domains, J. Exp. Med. 204 (6):1249-56). Thusboth aHUS and MPGN2 are due to subtle differences in Factor H control ofC3b breakdown.

FI knockout mice do not show C3b deposition on their glomeruli despitehaving all their plasma C3 converted to C3b because of unrestrainedaction of the C3b-feedback cycle. Mice with both FI and Factor Hdeficiency also do not develop MPGN2. However, injection of FI into thedouble k/o animals restores the MPGN2 pattern of C3 deposition in theglomeruli. This experiment demonstrates conclusively that conversion ofC3b to iC3b is absolutely required for the development of theinflammatory renal disease (K. L. Rose, D. Paixao-Cavalcante, J. Fish,A. P. Manderson, T. H. Malik, A. E. Bygrave, T. Lin, S. H. Sacks, M. J.Walport, H. T. Cook, M. Botto, and M. C. Pickering (2008), Factor I isrequired for the development of membranoproliferative glomerulonephritisin factor H-deficient mice, J. Clin. Invest. 2008 February; 118(2):608-18; January 17 [Epub ahead of print].

These disturbances of C3b feedback thus predispose to some relativelyacute renal conditions (such as aHUS and MPGN2) that occur in early lifeas well as more chronic conditions (e.g., AMD) that occur later in life.It, therefore, appears to be the case that the existence of agenetically determined systemic pro-inflammatory complement phenotypeallows progressive damage to occur in end-organs as a cumulative resultof multiple episodes of complement activation. This type of damage canoccur much earlier in life in the kidneys than it does in the eye.

AMD, Complement and Alzheimer's Disease: the possibility that theremight be some association between AMD and Alzheimer's disease was raisedby Dentchev et al. (T. Dentchev, A. H. Milam, V. M. Lee, J. Q.Trojanowski, and J. L. Dunaief (2003), amyloid-beta is found in drusenfrom some age-related macular degeneration retinas, but not in drusenfrom normal retinas, Mol. Vis. 9:184-190) who reported that β amyloidprotein could be found in the drusen from some AMD retinas but was notfound in drusen from normal retinas. They suggested that β amyloid, theprotein associated with the characteristic deposits in Alzheimer'sdisease might also play a role in AMD. This suggestion gained supportfrom the work of Yoshida et al. (T. Yoshida, K. Ohno-Matsui, S.Ichinose, T. Sato, N. Iwata, T. C. Saido, T. Hisatomi, M. Mochizuki, andI. Morita (2005), the potential role of amyloid beta in the pathogenesisof age-related macular degeneration, J. Clin. Invest. 115(10):2793-2800) who studied the effects of β amyloid protein on retinalpigment endothelial cells in vitro and showed the accumulation of thisprotein gave rise to some of the features characteristic of AMD,including retinal pigment epithelium atrophy and basal depositformation, as well as affecting the balance between VegF and PDF.

It was, therefore, of considerable interest when it was reported byZetterberg et al. (M. Zetterberg, S. Landgren, M. E. Andersson, M. S.Palmer, D. R. Gustafson, I. Skoog, L. Minthon, D. S. Thelle, A. Wallin,N. Bogdanovic, N. Andreasen, K. Blennow, and H. Zetterberg (2008),association of complement Factor H Y402H gene polymorphism withAlzheimer's disease, Amer. J. Med. Genet. Part B (NeuropsychiatricGenet.) 147B:720-726) that there was an association of the Factor HY402H allele (which carries an increased risk of AMD) also withAlzheimer's disease. However, the association with Alzheimer's diseasewas evident only in those individuals also carrying the ApoE4 allele,which is known to be a strong predisposing genetic influence forAlzheimer's disease. This was the first indication that a geneticpredisposition to AMD was also associated with Alzheimer's disease.

A possible mechanism by which β amyloid might alter complement functionin the eye was reported by Wang et al. (J. Wang, K. Ohno-Matsui, T.Yoshida, A. Kojima, N. Shimada, K. Nakahama, O. Safranova, N. Iwata, T.C. Saido, M. Mochizuki, and I. Morita (2008), altered function of FactorI caused by amyloid β: implication for pathogenesis of age-relatedmacular degeneration from drusen, J. Immunol. 181:712-720) who foundthat β amyloid was able to inhibit Factor I function. This intriguingobservation would benefit from some further studies to determine thestoichiometry of the reaction and whether the inhibition is competitive.Such inhibition of Factor I would provide a mechanism by which thedeposition of β amyloid protein at a local site would give rise to ahyperinflammatory effect by reducing the activity of the C3 breakdownpathway. Even more recently, it has been reported by Wang et al. (J.Wang, K. Ohno-Matsui, T. Yoshida, N. Shimada, S. Ichinose, T. Sato, M.Mochizuki, and I. Morita (2009), amyloid-β up-regulates complementFactor B in retinal pigment epithelial cells through cytokines releasedfrom recruited macrophages/microglia: another mechanism of complementactivation in age-related macular degeneration, J. Cel. Physiol.220:119-128) that β amyloid also upregulates the production of Factor Bin retinal pigment epithelium cells. It apparently does this byrecruiting microglia, which then produce cytokines that increase theproduction of Factor B. This would again produce a localhyperinflammatory state by increasing the activity of the C3b feedbackcycle.

These reports support the idea that β amyloid protein may play a role inthe pathogenesis of AMD by affecting the activities on the C3 feedbackand breakdown cycles, in both cases promoting the hyperinflammatoryphenotype. Also supporting such a connection is the finding thatantibodies to β amyloid protein attenuate disease in a mouse model ofAMD (J. D. Ding, J. Lin, B. E. Mace, R. Herrmann, P. Sullivan, and C. B.Rickman (2008), targeted age-related macular degeneration withAlzheimer's disease based immunotherapies: anti-amyloid-β antibodyattenuates pathologies in an age-related macular degeneration mousemodel, Vis. Res. 48:339-345). Further support for the role of theamplification loop in Alzheimer's disease is the very recent report ofLambert et al. (2009) (genome-wide association study identifies variantsat CLU and CR1 associated with Alzheimer's disease, Nat. Genet.September 6 (Epub ahead of print)) of whole genome associations inAlzheimer's disease, which showed a modest association (odds ratio 1.2195%, confidence interval 1.14-1.29) with CR1.

These findings on the effects of β amyloid protein on the complementamplification loop raise the possibility that these same mechanisms mayalso be at work in Alzheimer's disease and that here too it could beadvantageous to down-regulate the hyperinflammatory complementphenotype, particularly in those who carry the ApoE4 allele.

2. Therapeutic Considerations

The above analysis suggests that diseases such as aHUS and AMD might betackled by targeted intervention in the C3b feedback and iC3b generationpathways.

Raising the concentration of FI by infusion of purified enzyme is thepreferred strategy for so doing. It is known that quite modestaugmentation of Factor I concentration in human plasma strikinglyinhibits C3b feedback whether this is initiated by an alternativepathway activator (particulate inulin) or by a classical pathwayactivator (aggregated IgG) (P. J. Lachmann and L. Halbwachs (1975), theinfluence of C3b inactivator (KAF) concentration on the ability of serumto support complement activation, Clin. Exp. Immunol. 21:109).Furthermore, raising Factor I concentration will also accelerate iC3bbreakdown to C3dg and C3c and thereby reduce the inflammatory effectsdue to its reaction with CR3.

The alternative strategy of raising plasma Factor H concentrations byinfusion of this protein will also dampen feedback activity by providingmore cofactor activity for the conversion of C3b to iC3b (the “firstclip”) but will lead to an increased level of iC3b since it has noco-factor activity for the FI mediated breakdown of iC3b (the “secondclip”). Only the complement receptor CR1 (CD35) has this co-factoractivity in-vivo.

Inhibiting the amplifier enzyme Factor D provides a mechanism to reducethe formation of C3b but has no effect on the formation or breakdown ofiC3b. Genetic deficiency of Factor D in man does not give rise to kidneydisease

In terms of practical therapy, the Factor I strategy is also moreattractive because the plasma concentrations of FI are relatively low(equivalent to about 35 mg/litre in man) whereas addition of exogenousFactor H would require at least 10 times as much protein.Pharmacological inhibition of Factor D has been attempted (e.g., G. I.Glover et al., Mol. Immunol. 1988, 25:1261-7) and compounds based on 3,4dichloroisocoumarin or isatoic anhydride were found to be effectiveinhibitors of the enzyme but with insufficient selectivity to be viabledrugs (e.g., H. Jing et al., J. Mol. Biol. 1998, 282:1061-81).

This disclosure is, therefore, based on the therapeutic use ofrecombinant or plasma-derived FI. Early studies on addition of exogenousFI in experimental systems suggested that supplementation probablyneeded to increase blood levels by no more than 25% (Lachmann andHalbwachs (1975)). Based on the gene association studies noted above, itis likely that a chronic increase in FI plasma concentration of perhapsas little as 10% could have therapeutic effects if other mechanisticconditions were met. This would imply doses in man in the region of10-20 mg of protein administered systemically at intervals of severalweeks (see J. B. Ziegler, C. A. Alper, F. S. Rosen, P. J. Lachmann andL. Sherington (1975), restoration by purified C3b inactivator ofcomplement-mediated function in vivo in a patient with C3b inactivatordeficiency, J. Clin. Invest. 55:668).

In the case of aHUS, a plausible protocol would be to give a dose ofFactor Ito a genetically predisposed patient whenever he/she has aninfection or a fever of any cause. The same treatment would similarly begiven to any patient who has already had one attack of aHUS independentof their genotype.

In the case of AMD, it would probably be advisable to give the FIregularly every 2-4 weeks as soon as drusen have been detected or evenearlier where there is a genetic predisposition and a family history.

Subjects with evidence of atherosclerotic vascular disease that is moresevere than would be expected from the Framingham predictions could alsobenefit from damping down their complement system particularly if theyhave any of the genetic predispositions described above. In thisconnection the findings of the EPIC-Norfolk study that, in healthymiddle-aged subjects, a raised neutrophil count (a surrogate marker fora pro-inflammatory phenotype) is a predictor of earlier mortality (J. S.Rana, S. M. Boekholdt, P. M. Ridker, J. W. Jukema, R. Luben, S. A.Bingham, N. E. Day, N. J. Wareham, J. J. Kastelein, and K. T. J. Khaw(2007), differential leucocyte count and the risk of future coronaryartery disease in healthy men and women: the EPIC-Norfolk ProspectivePopulation Study Intern Med. 262 (6):678-89) are also significant. Thereshould be benefit in treating subjects in the top decile of neutrophilscounts prophylactically with FI as soon as they show any signs ofatherosclerotic vascular disease.

3. Specific Embodiments of the Disclosure

The large-scale isolation of the FI from Cohn Fractions 3 and 4 of humanplasma has been accomplished using affinity chromatography on columnscoupled with an anti-FI monoclonal antibody and may also be carried outusing other specific chromatography methods that have been applied toplasma products such as those employing dye ligands (see, for example,E. Gianazza and P. Arnaud (1982), a general method for fractionation ofplasma proteins, dye-ligand affinity chromatography on immobilizedCibacron blue F3-GA, Biochem J. 201:129-36). Factor I can be sterilizedby ultrafiltration/nanofiltration (T. Burnouf and M. Radosovich (2003),nanofiltration of plasma-derived biopharmaceutical products, Haemophilia24-37) and/or viral destruction in plasma feedstock (L. M. Williamsonand J. P. Allain (1995), virally inactivated fresh frozen plasma, VoxSang. 69:159-65) or heating of the purified product or concentrate (alsoK. L. and J. Kihl (1989), pasteurization of a factor I (C3b inactivator)concentrate from human plasma, Vox Sang. 57:240-2).

Production of recombinant human FI protein has also been reported frombaculovirus/insect cells (C. G. Ullman, D. Chamberlain, A. Ansari, V. C.Emery, P. I. Haris, R. B. Sim, S. J. Perkins (1998), human complementfactor I: its expression by insect cells and its biochemical andstructural characterization, Mol. Immunol. 35:503-12) and from COS andCHO cells. (M. J. Wong, G. Goldberger, D. E. Isenman, J. O. Minta(1995), processing of human factor I in COS-1 cells co-transfected withfactor I and paired basic amino acid cleaving enzyme (PACE) cDNA, Mol.Immunol. 32:379-87).

The factor I materials of this disclosure may be formulated intopharmaceutical compositions comprising a carrier suitable for thedesired delivery method. Suitable carriers include any material that,when combined with this protein, retains the function of the protein andis non-reactive with the subject's immune systems. Examples include anyof a number of standard pharmaceutical carriers such as sterilephosphate buffered saline solutions, bacteriostatic water, and the like(see, generally, Remington: The Science and Practice of Pharmacy, 2005(21st Edition, Popovich, N (eds), Advanced Concepts Institute,University of the Sciences in Philadelphia, Pa.).

One or more human factor I formulations may be administered via anyroute capable of delivering the protein to the disease site. Routes ofadministration include, but are not limited to, intravenous,intraocular, intraperitoneal, intramuscular, intradermal and the like.Factor I preparations may be lyophilized and stored as a sterile powder,preferably under vacuum, and then reconstituted in bacteriostatic watercontaining, for example, benzyl alcohol preservative, or in sterilewater prior to injection. Treatment will generally involve the repeatedadministration of the protein preparation via an acceptable route ofadministration such as intravenous (IV) or intraocular (IO) injection atan effective dose.

Dosages will depend upon various factors generally appreciated by thoseof skill in the art, including the route of administration, the type ofdisease and the severity, stage of the disease, the plasma half life ofthe protein in the preparation (the plasma half life of Factor I isbelieved to be approximately one week), the background factor I levelsin the patient, the desired steady-state protein concentration level,and the influence of any therapeutic agents used in combination with thetreatment method of the disclosure.

A typical normal plasma concentration of Factor I in man is about 35micrograms/ml. Allowing an extracellular fluid volume of about 10 litersgives a total of 350 mg Factor I. To raise this acutely by 10% wouldtake 35 mg; to raise it by 25% would take 88 mg. Preferred amounts ofFactor I are up to three times this amount, for example, up to 100-250mg every 1-3 weeks.

For IV or intramuscular administration, doses are likely to range fromabout 0.05 to 20 mg/kg with a frequency of dosing between daily andmonthly as repeated administrations may be required to achieve diseaseinhibition or regression. IO administration will involve significantlylower doses in the likely range of 1 to 1000 micrograms/eye. Adetermining factor in defining the appropriate dose is the amount of aparticular preparation necessary to be therapeutically effective in aparticular disease context and this can only be determined by clinicalinvestigation.

Patients may be evaluated for plasma factor I levels during therapy inorder to assist in the determination of the most effective dosingregimen and related factors. Conventional assay methods based onbreakdown of C3b or C4b in the presence of a cofactor may be used forquantifying circulating factor I in patients prior to or duringtreatment.

EXAMPLE The Influence of Factor I (FI) Concentration on the Ability ofSerum to Support Complement Activation Materials and Methods

Inulin—a standard suspension (50 mg/ml) in saline was sonicated.Dilutions of this suspension were made, the final concentration ofinulin in the serum being given for each experiment.

Aggregated human γ-globulins—these were obtained by heating aconcentrated solution (27 mg/ml) of human γ-globulins at 63° C. for 15minutes.

Factor I—functionally purified FI was prepared from the euglobulinfraction of serum by DEAE-cellulose and Sephadex G-200 chromatography(Lachmann, Aston and Nicol, 1973). The titer of the purified FI standardsolution was measured and compared to the normal serum titer by itscapacity of inducing EAC143 agglutination in presence of bovineconglutinin (Lachmann, and Muller-Eberhard, 1968). Variations in FIconcentration in normal human serum were obtained by adding differentdilutions of the purified FI standard solution to the serum.

Effect of Increased FI Concentration on Complement Activation by Inulin(FIG. 3).

FI inhibits C3 conversion by inulin at all concentrations of inulinused. It also inhibits factor B conversion by inulin. Quite smallamounts of FI are sufficient for this inhibitory effect: an increase ofonly 15% of the normal FI concentration in the serum results in 50%inhibition of C3 conversion by inulin.

Effects of Increased FI Concentration on Complement Activation byAggregated γ-Globulins (FIG. 3).

C3 conversion by aggregated γ-globulins is also by increased FIconcentration, but in this case more than 20% increase is necessary inorder to observe the inhibition.

CONCLUSION

It is clear from the experiments described that the FI concentration inwhole human serum is by no means so high that further elevation has noeffect on complement activities. In fact, quite modest increases in theFI concentration (15-25%) markedly inhibit the capacity of a typicalproperdin pathway activator like inulin and (to a slightly lesserextent) of a typical classical pathway activator like aggregated humanIgG to produce complement activation. This evidence suggests thatvariations in FI concentration even within physiological limits maysignificantly modulate complement activation.

REFERENCES

Lachmann, P. J., W. P. Aston, and P. A. E. Nicol (1973), Immunochemistry10:695;

Lachmann, P. J., and H. J. Muller-Eberhard (1968), J. Immunol. 100:691.

1-73. (canceled)
 74. A method of lowering the amount of C3b activity ina subject, the method comprising: increasing the level ofC3b-inactivating activity and/or iC3b degrading activity in the subject;wherein the level of C3b-inactivating activity and/or iC3b degradingactivity in the subject is increased to a level that exceeds a normallevel of C3b-inactivating activity and/or iC3b degrading activity in thesubject.
 75. The method according to claim 74, further comprising notincreasing the level of Factor H in the subject.
 76. The methodaccording to claim 74, wherein increasing the level of C3b-inactivatingactivity and/or iC3b degrading activity in the subject comprisesincreasing the level of Factor I in the subject.
 77. The methodaccording to claim 76, wherein the Factor I has at least 90% amino acididentity with native Factor I; and wherein the Factor I retainsC3b-inactivating activity and/or iC3b degrading activity.
 78. The methodaccording to claim 74, wherein the level of C3b-inactivating activityand/or iC3b degrading activity is increased by at least 10% above thenormal level.
 79. The method according to claim 74, wherein the level ofC3b-inactivating activity and/or iC3b degrading activity is increased byno more than 50% above the normal level.
 80. The method according toclaim 74, wherein the subject has a normal level of C3b-inactivatingactivity and/or iC3b degrading activity prior to increasing the level ofC3b-inactivating activity and/or iC3b degrading activity in the subject.81. The method according to claim 74, wherein the subject is human. 82.The method according to claim 74, wherein the subject suffers fromAge-related Macular Degeneration or atypical hemolytic uremic syndrome.83. The method according to claim 74, wherein the level ofC3b-inactivating activity and/or iC3b degrading activity in the plasmaof the subject is increased to a level that exceeds a normal level ofC3b-inactivating activity and/or iC3b degrading activity in the plasmaof the subject.
 84. The method according to claim 83, wherein the normallevel is in the range of 30-40 μg/ml Factor I in the plasma of thesubject.
 85. The method according to claim 74, wherein the level ofC3b-inactivating activity and/or iC3b degrading activity in an eye ofthe subject is increased to a level that exceeds a normal level ofC3b-inactivating activity and/or iC3b degrading activity in an eye ofthe subject.